A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model

Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Ablation of xeno-reactive antigens, especially the 1,3-Gal epitope (GalT), which causes hyperacute rejection, and insertion of complement regulatory protein genes, such as hCD46, hCD55, and hCD59, and genes to regulate the coagulation pathway or immune cell-mediated rejection may be required for an ideal xenotransplantation model. However, the technique for stable and efficient expression of multi-transgenes has not yet been settled to develop a suitable xenotransplantation model. To develop a stable and efficient transgenic system, we knocked-in internal ribosome entry sites (IRES)-mediated transgenes into the α 1,3-galactosyltransferase (GGTA1) locus so that expression of these transgenes would be controlled by the GGTA1 endogenous promoter. We constructed an IRES-based polycistronic hCD55/hCD39 knock-in vector to target exon4 of the GGTA1 gene. The hCD55/hCD39 knock-in vector and CRISPR/Cas9 to target exon4 of the GGTA1 gene were co-transfected into white yucatan miniature pig fibroblasts. After transfection, hCD39 expressed cells were sorted by FACS. Targeted colonies were verified using targeting PCR and FACS analysis, and used as donors for somatic cell nuclear transfer. Expression of GalT, hCD55, and hCD39 was analyzed by FACS and western blotting. Human complement-mediated cytotoxicity and human antibody binding assays were conducted on peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs), and deposition of C3 by incubation with human complement serum and platelet aggregation were analyzed in GGTA1 knock-out (GTKO)/CD55/CD39 pig cells. We obtained six targeted colonies with high efficiency of targeting (42.8% of efficiency). Selected colony and transgenic pigs showed abundant expression of targeted genes (hCD55 and hCD39). Knocked-in transgenes were expressed in various cell types under the control of the GGTA1 endogenous promoter in GTKO/CD55/CD39 pig and IRES was sufficient to express downstream expression of the transgene. Human IgG and IgM binding decreased in GTKO/CD55/CD39 pig and GTKO compared to wild-type pig PBMCs and RBCs. The human complement-mediated cytotoxicity of RBCs and PBMCs decreased in GTKO/CD55/CD39 pig compared to cells from GTKO pig. C3 was also deposited less in GTKO/CD55/CD39 pig cells than wild-type pig cells. The platelet aggregation was delayed by hCD39 expression in GTKO/CD55/CD39 pig. In the current study, knock-in into the GGTA1 locus and GGTA1 endogenous promoter-mediated expression of transgenes are an appropriable strategy for effective and stable expression of multi-transgenes. The IRES-based polycistronic transgene vector system also caused sufficient expression of both hCD55 and hCD39. Furthermore, co-transfection of CRISPR/Cas9 and the knock-in vector not only increased the knock-in efficiency but also induced null for GalT by CRISPR/Cas9-mediated double-stranded break of the target site. As shown in human complement-mediated lysis and human antibody binding to GTKO/CD55/CD39 transgenic pig cells, expression of hCD55 and hCD39 with ablation of GalT prevents an effective immunological reaction in vitro. As a consequence, our technique to produce multi-transgenic pigs could improve the development of a suitable xenotransplantation model, and the GTKO/CD55/CD39 pig developed could prolong the survival of pig-to-primate xenotransplant recipients.

www.nature.com/scientificreports/ using hCD39 expression was performed to select targeted cells because we expected that only targeted cells would express hCD39. hCD39 positive cells were 20% of the total cells and sorted (supplementary Fig. 1C). After selection using FACS sorting, 24 single colonies were picked and 14 colonies were analyzed to verify targeting through targeting PCR (supplementary Fig. 1D). In six colonies the vector was successfully targeted on exon 4 of the GGTA1 gene with high efficiency (42.8% of efficiency) ( Table 2). Colony #15 was used as donor for somatic nuclear transfer (SCNT).
Ablation of GalT and GGTA1 endogenous promoter-mediated expression of hCD55 and hCD39. We selected one of the targeted six colonies, which had greater cell morphology and proliferation.
hCD55 and hCD39 were abundantly expressed on the cell surface of targeted donor cells and expression of the proteins was also confirmed (supplementary Fig. S1E and S1F). The targeting vector was knocked-in to the one allele of the GGTA1 locus, with consequent heterozygous knock-out of the GGTA1 gene. However, the other allele which was not knocked-in was also disrupted by CRISPR/Cas9, thereby inducing homozygous knock-out of the GGTA1 gene ( Fig. 1A and supplementary Fig. S4C).

Production of GTKO/CD55/CD39 knock-in targeted transgenic cloned pig. Cloned piglets pos-
sessing the targeting vector inserted into the GGTA1 locus were produced and their genotype verified by targeting PCR (Fig. 1B). hCD55 and hCD39 was abundantly expressed in ear skin fibroblasts cells, aorta endothelial cells, cornea endothelial cells, peripheral blood mononuclear cells (PBMCs), red blood cells (RBCs), and kidney cells from GTKO/CD55/CD39 transgenic pig. The transgenic animals also revealed ablation of the aGal epitope on the surface of these cells (Fig. 1C). Moreover, the expression level of hCD55 was similar between human and GTKO/CD55/CD39 pig aorta endothelial cells. In the case of hCD39, the expression level in GTKO/CD55/ CD39 pig aorta endothelial cells was higher than in human endothelial cells (Fig. 1D).
Human antibody binding. Human antibody binding to PBMCs and RBCs from GTKO/CD55/CD39 pigs was greatly reduced compared with binding to PBMCs and RBCs from wild-type pigs. However, human antibody binding to PBMCs ( Fig. 2A) and RBCs (Fig. 2B) was not significantly different between GTKO and GTKO/ CD55/CD39 pig.

Inhibition of immunological rejection by human complement.
The viability against human complement was estimated using peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs) from wild-type, GTKO, and GTKO/CD55/CD39 pig. Both GTKO/CD55/CD39 cell types remarkably prevented human complement-mediated cytotoxicity against various human complement serum concentration (0%, 3.125%, 6.25%, 12.5%, and 25%) compared with wild-type and GTKO cells. Against most concentrations of human complement serum, the viability of PBMCs and RBCs from GTKO/CD55/CD39 pig was not significantly different from that of human PBMCs or RBCs. As shown in Fig. 2C Platelet aggregation. Platelet aggregation was tested in wild-type and GTKO/CD55/CD39 transgenic pigs' PRP.

Discussion
Production of transgenic pigs is an imperative technology for the success of pig-to-human xenotransplantation. Many genes have to be engineered to overcome various immunological rejection issues in pig-to-human xenotransplantation. Many transgenic pigs with single gene or multi-transgenic pigs with more than three engineered genes were developed in the xenotransplantation field 30,33,55,56 . Recently, the successful first clinical heart and kidney xenotransplantation 57 was carried out using transgenic pig with ten genetic modification. Nevertheless, an effective transgenic system has not been agreed upon. In our previous study 52 , we chose the GGTA1 locus and a gene trap strategy for transgene expression due to stable and various expression of GalT [58][59][60][61][62] . As a result, our transgenic pig expressed the transgene abundantly without an exogenous promoter, and the transgene was well maintained in expression for several generations. Therefore, we tried to produce multi-transgenic pigs by a gene trap strategy targeting the GGTA1 locus, for the stable and efficient expression of transgenes to solve the issues with exogenous promoter-mediated transgene expression and random integration of transgenes. The homologous recombination strategy is an effective method for gene modification in mouse embryonic stem cells by knock-out and knock-in 63,64 . Since gene-editing technologies such as ZFN, TALEN, and CRISPR/ Cas9 have been developed, some studies have attempted to use TALEN or CRISPR/Cas9 systems to increase the efficiency of homologous recombination-mediated knock-in [65][66][67][68] . In this sense, we developed a system that combined CRISPR/Cas9 and a hCD55/hCD39 knock-in vector to increase efficiency. We chose to target exon 4, which includes the initiation codon of the GGTA1 gene, so that expression of hCD55 and hCD39 would be under the control of the GGTA1 gene endogenous promoter, due to the wide expression of GalT in pig, and to induce the stable expression of the transgene. We constructed a targeting vector to ablate the GGTA1 gene and express hCD55 and hCD39 by the gene trap principle. The combination of CRISPR/Cas9 and knock-in systems increased the frequency of homologous recombination, so that the targeted gene was detectable by FACS sorting (20% expression of hCD39 after neomycin selection, supplementary Fig. 1C). After FACS sorting, we obtained six precise targeted colonies of a total of 20 colonies analyzed (42.8% efficiency), with a much higher efficiency than in our previous study (2.6% of efficiency) 52 (Table 2). Furthermore, another allele apart from the targeted allele was also mutated by the CRISPR/Cas9 effect, whereas knock-in usually resulted in heterozygous targeting ( Fig. 1A and supplementary Fig. S4C).
Anti-GalT antibodies are naturally developed in human in the first few months of infancy [69][70][71] . It is probably due to the fact that the infant is exposed to viruses or microorganisms expressing GalT in the gastrointestinal tract 72 . Because of this phenomenon, GalT expressed on the cell surface in pigs is the main hurdle causing hyperacute rejection in pig-to-human xenotransplantation. Antigen-antibodies mediated rejection still remains the main cause for xenotransplantation failure following knock-out of GalT. Other non-gal antigens, such as Neu5Gc or Sda, have been knocked out to decrease anti-pig antibody binding in human 9,73,74 . However, the survival of Neu5Gc deficient pig kidney was shorter than GTKO pig kidney in pig-to-nonhuman primate xenotransplantation, and antibody binding to Neu5Gc deficient pig were also increased in old world monkey because unlike humans, old world monkey have activated CMAH gene, and possess naturally-existing antibodies directed to Neu5Gc deficient pig [74][75][76] . Therefore, the current model of pig-to-non-human primate xenotransplantation is less than ideal given the expectation for improved survival in the first pig-to-human trials with a Neu5Gc deficient pig compared to the pig-to-non-human primate model. These findings and our results imply that triple knockout of GGTA1, CMAH and β4GalNT2 and insertion of hCD55 and hCD39 genes could be effective to prevent immune rejection in pig-to-human xenotransplantation.
hCD55 plays a role in the regulation of complement activation, which is caused by the antigen-antibody complex via the classical pathway. The mechanism between hCD55 and NK cells is unknown, however hCD55 was associated with NK cell-mediated lysis, whereas other complement regulatory proteins including hCD46 and hCD59 were not 22 . In this study, we successfully eliminated the GalT epitope through targeting of the hCD55/ hCD39 knock-in vector into the GGTA1 gene locus. Ablation of the GalT epitope considerably decreased human complement-mediated cytotoxicity and human antibody binding compared to wild-type pig cells. Although human antibody binding to cells from GTKO/CD55/CD39 pig was not greater than to cells from GTKO pigs, our transgenic pig cells showed further prevention against human complement-mediated lysis compared to GTKO pig cells (Fig. 2).
Decrease of C3 deposition on GTKO/CD55/CD39 pig cells following activation of the complement cascade was comparable to that in cells from GTKO, that also proved the inhibitory effect of the complement cascade by targeting hCD55 (Fig. 3A).
These data imply that the hCD55 gene targeted in our GTKO/CD55/CD39 pig could be capable of dramatically controlling the complement reaction in pig-to-human humoral rejection. Although precluding hyperacute www.nature.com/scientificreports/ rejection or complement activation, coagulation disorders also remain a barrier for successful pig-to-primate xenotransplantation. At the sites of vascular injury, platelet adhesion is initiated and activated by agonists, www.nature.com/scientificreports/ including ADP and collagen. Therefore, platelets, which have a role in the interaction between endothelium and leukocytes in the form of platelet microthrombi, have been thought to be a major player in the process of improving survival of organ xenotransplantation 77,78 . To regulate platelet-mediated thrombosis, we also aimed to knock-in hCD39 with hCD55. As a result, the platelet aggregation was much more delayed in PRP from GTKO/ CD55/CD39 pig than PRP from wild-type and GTKO pigs, for not only ADP-induced, but also collagen-induced platelet aggregation (Fig. 2B). These results showed that our GTKO/CD55/CD39 pig could prevent not only humoral rejection but also platelet-mediated thrombosis in pig-to-primate xenotransplantation. Our endogenous-mediated expression of transgenes by knock-in, targeting of hCD55 and hCD39 into the GGTA1 locus, induced abundant and stable expression of functioning proteins as well. Ablation of the GalT epitope and concomitant expression of hCD55 improved the inhibition of complement-mediated cytotoxicity compared to wild-type and GTKO pigs. The function of hCD39 was also proven by platelet aggregation analysis. Although hCD55 and hCD39 are not novel genes for xenotransplantation, endogenous promoter-mediated expression of hCD55 and hCD39 in a transgenic pig had not been tried in pig-to-non-human primate xenotransplantation. Moreover, our transgenic pig showed similar expression levels of hCD55 and higher expression of hCD39 compared to human aorta endothelial cells (Fig. 1D). In this phenomenon, our transgenic pig could be a comparable model to various transgenic pigs for xenotransplantation.

Methods
Ethical statements and animal carep. All experimental methods were carried out according to ARRIVE guidelines. All methods were performed in accordance with relevant guidelines and regulations. All protocols of animal care and use procedure were approved by the Institutional Animal Care and Use Committee of Optipharm, Inc., Life Science Institute (IACUC approval No. OPT-140103-1, D-grade). All pigs used in the experiments were white Yucatan miniature pigs and approved by institutional animal care. The animal facility for all pigs at Optipharm, Inc., was a specific pathogen-free environment. Filtered water and air, and sterilized feed were supplied, and the rooms were maintained at 24 °C ± 2 °C and 12 h light/12 h dark cycles. For experiments of this study, one-year-old healthy pigs, wild-type, GTKO, and GTKO/CD55/CD39 were humanely euthanized by intravenous injection of 2 mM/kg potassium chloride solution under general anesthesia. After a veterinarian certified the death of the pigs, their organs were harvested.

Construction of hCD55/hCD39 knock-in vector and CRISPR/Cas9 vector for GalT. Two homolo-
gous arms (left and right arms) were designed to target exon4 of the GGTA1 gene as described in Fig. 1. These arms were amplified from pig genomic DNA by PCR. hCD55 and hCD39 fragments were synthesized from human cDNA by PCR. Left and right arm DNA fragments were inserted into modified PGKneolox2DTA.2 . Experiments were performed in triplicated (n = 1, ***P < 0.05, one-way analysis of variance). Inhibition of human complement mediated rejection on cells from GTKO/CD55/CD39 pig. PBMCs (C) and RBCs (D) from wild-type, GTKO/CD55/CD39 pig and human. These cells were incubated with various human complement serum concentration, and the viability was represented by calculation with absorbance of CCK-8 (PBMCs) or viable remaining cells (RBCs). Experiments were performed in triplicated (n = 1; * P < 0.05; **P < 0.01; ***P < 0.001; ns, non-significant; two-way analysis of variance). www.nature.com/scientificreports/ (Addgene plasmid 13449). hCD55 and hCD39 fragments were inserted between the left arm and the neomycin fragment. IRES sequences were inserted between the hCD55 and hCD39 fragments to express hCD39. Small guide RNA sequences (5'-AAT GAA TGT CAA AGG AAG AG-3') for CRISPR/Cas9 vector were targeted for the ATG codon of the GGTA1 gene.
Production of cloned pig from SCNT and embryo transfer. SCNT and embryo transfers were performed as described previously in our study 37 . The pregnancy status was monitored with an ultrasound scanner (Mysono 201, Medison Co., LTD, Korea).

Verification of targeted donor cells and transgenic pigs. From the single cell culture, 24 colonies
were picked and 20 colonies were expanded to be analyzed. Each colony was harvested in 12-well plate culture vessels. Genomic DNA was extracted using DNeasy blood and tissue kit (QIAGEN, USA) from each colony and cloned pig tissue. Left arm, right arm, and long PCR amplification were performed to screen targeted colony and cloned pig using primers as described in supplementary Table S1. in 100 µL of Dulbecco's phosphate buffered saline (DPBS, Gibco) and stained with 2 µL of anti-hCD55 (sc-57133, Santacruz) or anti-hCD39 (sc-65232, Santacruz) for 2 h at room temperature. Fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were then incubated for 1 h on ice to analyze the expression of hCD55 and hCD39. To analyze GalT expression, 2 µL of Alexa Flour 488®-conjugated isolectin-ib4 (I21411, Invitrogen, USA) was incubated with the suspended cells for 3 h on ice. Expression of GalT, hCD55, and hCD39 on the cell surface was estimated by flow cytometry (Calibur-S system, BD). To identify translational expression of hCD55 and hCD39, 2 × 10 6 cells were harvested and lysed with 30 µL of mammalian protein extract reagent (Thermo, USA) for 4 h on ice. Proteins extracted from the cells were quantified using the Bradford assay (Bio-Rad, USA) and resolved on 8% sodium dodecyl sulfate polyacrylamide gels. The proteins were then transferred to nitrocellulose membrane, which was blocked with 1X Tris-buffered saline, 0.1% Tween-20 containing 5% skimmed milk for 1 h at room temperature. Incubation with anti-hCD39 (sc-33558, Santacruz), or anti-hCD55 (ab54595, Abcam), or anti-beta-actin (sc-47778, Santacruz) as primary antibodies happened overnight at 4 °C. The membrane was then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Human hCD55 and hCD39 were detected with the ECL chemiluminescence kit for western blot analysis (GE Healthcare, UK), according to the manufacturer's protocol.

Isolation of PBMCs and RBCs.
A total of 5 mL of whole blood from wild-type, GTKO (developed in our previous study 79 ), and GTKO/CD55/CD39 pigs was collected into EDTA tubes (BD). PBMC and RBCs were separated from collected blood using density media ficoll-paque plus (GE), according to manufacturer's protocol. Isolated PBMCs and RBCs were washed with DPBS (Gibco), and immediately used for complementmediated cytotoxicity or human antibody binding assays, or for confirmation of gene expression.

Culture of Aorta endothelial cells, corneal endothelial cells, and kidney cells.
Pig aortas were incubated with 0.005% collagenase type IV (sigma, USA), and the isolated cells were washed with DPBS(Gibco). The cells were then culture with endothelial cell growth medium-2 bullet kit (Lonza, Switzerland). Pig corneal endothelial cells were isolated and cultured, as described in our previous study 80 . Pig kidney tissues were minced into 1-2-mm sections, and digested with 3% collagenase type IV. The digested tissues were passed through a 100-m cell strainer (BD). The isolated cells were washed with DPBS (Gibco), and cultured in DMEM (Welgene) containing 15% FBS (Hyclone), 1% non-essential amino acid, 0.1 mM β-mercaptoethanol, and 1% antibioticantimycotic (Gibco).
Complement-mediated cytotoxicity. 2 × 10 5 of PBMCs and RBCs isolated from pigs were incubated with 0%, 3.125%, 6.25%, 12.5%, and 25% of pooled human complement serum (Innovative research, USA) for 2 h (PBMCs) or overnight (RBCs) on v-bottom 96-well plates (Corning, USA). After incubation, human complement serum was removed following centrifugation. PBMCs were then re-suspended with 100 µL of RPMI 1640 media (Gibco) containing 5% fetal bovine serum (Gibco), 15 mM HEPES (Sigma), and 0.2 M EDTA (Bioneer, Korea). To estimate viability against human complement serum, 10 µL of CCK-8 solution was added to suspended PBMCs, and the absorbance of each well at 450 nm was measured. After incubation of RBCs with human complement serum, viability was estimated by measurement of the absorbance (405 nm) of the remaining viable RBCs.
Human antibody binding assay. Pig PBMCs and RBCs were incubated with 10% normal human serum (Millipore, USA) for 30 min at 4 °C. PBMCs and RBCs were then washed by centrifugation and incubated with 100 μL of PBS containing 3 µL of anti-human IgG (62-8411, Invitrogen) or anti-human IgM (F5384, Sigma) for 1 h at 4 °C. Stained PBMCs and RBCs were analyzed using flow cytometry (CantoII, BD). Human antibody binding was calculated using the MFI formula below: C3 deposition. RBCs from wild-type, GTKO, and GTKO/CD55/CD39 pigs (5 × 10 5 ) were incubated with human complement serum (Innovative technology, USA) at a concentration range of 0%, 25%, and 50% for 24 h. The cells were then incubated with an anti-C3 antibody (Santacruz) for 2 h at room temperature. The FITCconjugated secondary antibody (Abcam) was applied for 1 h on ice. After each incubation, the cells were washed three times with 1 mL of PBS. Stained cells were analyzed by flow cytometry (CantoII, BD, USA).

Data availability
The datasets used and analysed during the current study available from the corresponding author on reasonable request.
Relative MFI = MFI of antibody stained with human serum incubation MFI of antibody stained cells without human serum incubation